PROJECT SUMMARY The critical preliminary data for this proposal is that in preclinical models, pre-existing tumor-resident T cells are both necessary and sufficient for tumor control by radiation therapy and immunotherapy. We demonstrate that in poorly immunogenic tumors, cross-presenting dendritic cells in the tumor-draining lymph node may be playing no significant role in tumor control. In these models, radiation and immunotherapy are an effective means of local control but fail to engender new systemic immunity. The aim of this proposal is to understand how radiation interacts with tumor resident T cells for local control, and how to restore cross-presentation of tumor-associated antigens to generate new systemic anti-tumor immune responses. We hypothesize that the endogenous vaccine effect of radiation therapy is limited in poorly immunogenic tumors, and current success relies on amplifying pre-existing anti-tumor immunity. The specific aims of this study are to 1: Test the hypothesis that in the presence of pre-existing immunity, tumor control by radiation therapy and immunotherapy occurs independent of cross-presentation and new immune responses; 2: Test the hypothesis that deficient DC cross-presentation limits the ability of radiation therapy to initiate new systemic anti-tumor immune responses; 3: Test the hypothesis that regulation of antigen presentation and cross-presenting DC can be assessed in patient tumors using genetic analysis. Our study design incorporates CT-guided radiation therapy and results are validated in multiple tumor models including those from genetically engineered spontaneous models, using a range of RT doses and fractionations. Our analyses of clinical samples use high quality bioinformatic approaches that allow us to evaluate the infiltrating immune cells and antigen presentation in patient tumors. These are applied to a unique clinical study that allows us to validate our preclinical observations in patients.